Stable engineered vascular networks from human induced pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels

Abstract

Here, we describe an in vitro strategy to model vascular morphogenesis where human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) are encapsulated in peptide-functionalized poly(ethylene glycol) (PEG) hydrogels, either on standard well plates or within a passive pumping polydimethylsiloxane (PDMS) tri-channel microfluidic device. PEG hydrogels permissive towards cellular remodeling were fabricated using thiol-ene photopolymerization to incorporate matrix metalloproteinase (MMP)-degradable crosslinks and CRGDS cell adhesion peptide. Time lapse microscopy, immunofluorescence imaging, and RNA sequencing (RNA-Seq) demonstrated that iPSC-ECs formed vascular networks through mechanisms that were consistent with in vivo vasculogenesis and angiogenesis when cultured in PEG hydrogels. Migrating iPSC-ECs condensed into clusters, elongated into tubules, and formed polygonal networks through sprouting. Genes upregulated for iPSC-ECs cultured in PEG hydrogels relative to control cells on tissue culture polystyrene (TCP) surfaces included adhesion, matrix remodeling, and Notch signaling pathway genes relevant to in vivo vascular development. Vascular networks with lumens were stable for at least 14 days when iPSC-ECs were encapsulated in PEG hydrogels that were polymerized within the central channel of the microfluidic device. Therefore, iPSC-ECs cultured in peptide-functionalized PEG hydrogels offer a defined platform for investigating vascular morphogenesis in vitro using both standard and microfluidic formats.

Human iPSC-derived endothelial cell sprouting assay in synthetic hydrogel arrays

“…In contrast to natural ECMs, PEG hydrogels are biologically inert[67], synthetic polymer networks that can be readily polymerized and functionalized to provide critical signals to ECs, including cell adhesion ligands[24,43,68] and protease-degradable linkers[24,37,68,69]. The parameters of PEG hydrogels including cell adhesion, stiffness, and degradability can be independently tuned for promoting particular cell functions[24,31,68]. Finally, thiol-ene photopolymerization enables rapid generation of cell-dense hydrogel spheres and hydrogel over-layers and is amenable to enhanced throughput 3D culture.…”

“…We designed a chemically-defined, quantitative assay of angiogenic sprouting behavior using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) that were previously shown to exhibit stable angiogenic phenotype and function and vascular network formation in vitro [23,24]. Thiol-ene photopolymerization was used to rapidly encapsulate iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres that were then embedded in a cell-free over-layer.…”

Biomaterials control of pluripotent stem cell fate for regenerative therapy

“…The MMP acted as a cross-linker between the different PEG chains, while the RGD sequence was incorporated to increase cell adhesion. Human iPSCs encapsulated in the gel matrix, as compared to those cultured on 2D dish, were able to develop vasculogenesis more profoundly through the upregulation of matrix remodeling and Notch signaling pathways, which was also found in the vasculature development in vivo [255]. Among the PEG hydrogels with different MMP cross-linking ratios, the intermediate MMP cross-linking showed an optimum vascularization, implying the importance of an appropriate matrix density and cell-induced degradation for vasculogenesis.…”

3D Printing of Tissue Engineered Constructs for In Vitro Modeling of Disease Progression and Drug Screening

“…85 A liver-like model utilizing an immortalized hepatoma cell line and ECM-derived hydrogel has been reported to not only recover hepatocyte function in 3D but also provide a means of high-throughput screening of drugs to assess liver toxicity in vitro . 67 Other in vitro models have been developed for organs including bone 4 , myocardium 16 , trachea 56 , vessels 89 , nerves 51 , cartilage 17 , and cornea. 1 …”